Our long-term goals are to develop clinically safe human butyrylcholinesterase (huBuChE) variants as hydrolytic catalysts to protect populations at risk from exposure to chemical warfare agents or organophosphate (OP) pesticides. The challenge to convert huBuChE into an OP hydrolytic catalyst is to identify huBuChE variants that are resistant to inactivation, catalyze OP hydrolysis efficiently and rapidly and spontaneously dephosporylate. The underlying hypothesis for our work is that combinations of huBuChE mutations at multiple residues, close to and/or far from the active site, are needed to markedly improve the OP hydrolysis activity to ultimately serve as a clinically useful hydrolytic catalyst. The primary goal of the current work is to apply powerful molecular evolution strategies to produce huBuChE variants with significantly improved hydrolytic activity for OP nerve agents. The Aims for this research include: 1) Stereoselective synthesis of different classes of enantiomerically pure OP model substrates for functional screening;2) Optimization and validation of the functional screening system with a site-saturation mutation library;3) Evolution of huBuChE variants with catalytic activity using two model compounds representing tabun and soman;4) Evolution of huBuChE variants with broad specificity;and 5) In vitro and in vivo efficacy testing of evolved huBuChE candidates using authentic nerve agents in collaboration with the Center. This work provides the research and development (R&D) effort required to identify OP catalytic huBuChE variants through a combinatorial approaches including molecular evolution, rational designed mutagenesis, in vitro and in vivo efficacy functional screening. Upon completion of this study, we will have the third generation huBuChE products ready to enter advanced development including production under good manufacturing practice (GMP) conditions, advanced pharmacological screening, and preclinical testing, a process that has been successfully established to transition two generations of huBuChE based protein drugs, human plasma derived huBuChE and recombinant wild-type huBuChE, for advanced product development.